1. Field of the Invention
The present invention relates generally to antibodies to Rh(Rhesus) factors which are present on the surface of red blood cells. More particularly, the invention is directed to improved Rh antibodies which may be used in Rh typing methods or as therapeutic agents.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography.
Rh factors are genetically determined antigens (agglutinogens) which are present on the surface of red blood cells. Rh factors form a complex blood group system which is of major importance in clinical medicine. The Rh(D) antigen is one of the most immunogenic and best known Rh factors. The presence or absence of Rh(D) antigen is especially important in blood transfusions because mixing of Rh(D) positive and Rh(D) negative blood types can result in blood clotting and other severe problems for the blood recipient. Rh(D) blood type is also important in pregnancy where severe problems can occur when the mother and fetus have Rh(D) blood types which do not match.
A number of methods have been developed to determine Rh blood type. The majority of the procedures are hemagglutination tests which utilize one or more reagents that include antibodies to Rh antigens. Typically, the reagents are added to a sample of red blood cells in a test well and incubated for a set period of time. The presence of Rh antigen is most often determined macroscopically by agglutination of the cells in the same manner as other conventional blood typing procedures.
Several modalities and antibody reagents are currently available to perform Rh hemagglutinin tests. In one type of test protocol, IgG anti-Rh antibodies are initially added to the red blood cells. IgG antibodies are “incomplete” antibodies with respect to the Rh antigen and therefor are not able to agglutinate the red blood cells by themselves. As a result, potentiating agents such as non-specific proteins or secondary anti-human IgG must be added to induce a positive agglutination reaction. This type of test is prone to false positives due to the non specific nature of the added potentiating agent.
In another type of Rh typing protocol, IgM anti-Rh antibodies are used in place of the IgG antibody. IgM is a complete antibody with respect to Rh and is capable of agglutinating Rh positive cells. However, IgM antibodies are not nearly as reactive as IgG antibodies. This results in the need for extended incubation times and the potential for false negative tests.
The basic Rh hemagglutination tests have been modified in a number of different ways in order to simplify the tests and make them more accurate. For example, a “gel test” has been proposed which uses a recombinant anti-Rh(D)Fab/phage and anti-M13phage antibody as the secondary reagent. Alternatively, IgG anti-D has been pretreated with reducing agents to open the hinge region of the molecule. Also, red blood cells have been treated with certain enzymatic preparations, such as papain, to render the cells susceptible to direct agglutination with IgG anti-Rh(D). Exemplary Rh(D) blood typing procedures are set forth in Reference Nos. 1-11.
Although the existing procedures for determining Rh blood type are well-suited for their intended purpose, there is still room for improvement. Accordingly, there is a continuing need to develop new reagents and protocols which will make Rh phenotyping even easier, faster and more accurate.
Rh(D) antibodies are also used as prophylactic agents. For example, the administration of Rh(D) antibodies for preventing the hemolytic disease of the newborn due to Rh incompatibility (HDN-Rh) is a very effective, and well established medical practice. HDN-Rh occurs to Rh(D) positive fetuses carried by previously immunized, Rh(D) negative, pregnant women. Rh(D) antibodies of the immunoglobulin G (IgG) class existing in the maternal blood cross the placental barrier and reach the fetal circulation. Once there, Rh(D) antibodies bind to and induce lysis of red blood cells, causing the disease. Since 1961 it is known that the administration of Rh(D) antibodies to pregnant women just after delivery prevents Rh(D) sensitization. More recently and based on accumulated evidence of Rh immunization during pregnancy, antenatal administration of Rh(D) antibodies (at 28 week's gestation) has also been proposed, and adopted in some countries. Despite its efficacy, the mechanism of action of the administered Rh(D) antibodies is not completely understood.
At the time of introduction of the Rh prophylaxis programme, the Rh(D) immunoglobulin for injection was derived mainly from the post-partum plasma of women immunized by pregnancy. Due to the success of this treatment the diminishing number of women immunized during pregnancy has reduced the availability of plasma-derived anti-D. This shortage lead to the recruitment and immunization of D negative male volunteers for the procurement of anti-D. Currently, anti-D used in prophylaxis is composed of immunoglobulin obtained almost entirely from these two sources.
Several considerations suggest that additional, non-plasma derived, sources of anti-D will be required in the near future. Firstly, the demand is expected to increase as the population grows. Secondly, the number of immunized, pregnant women donors is expected to diminish even more due to the efficacy of the Rh prophylaxis. Thirdly, immunization of D negative volunteers has been criticized due to potential risks of contamination with pathogenic, blood-borne viruses. An alternative approach to producing human anti-D is the monoclonal and recombinant antibody technologies.